design of selective, atp-competitive inhibitors of akt

pubs.acs.org/jmcPublished on Web 05/19/2010r 2010 American Chemical Society

J. Med. Chem. 2010, 53, 4615–4622 4615

DOI: 10.1021/jm1003842

Design of Selective, ATP-Competitive Inhibitors of Akt†

Kevin D. Freeman-Cook,* Christopher Autry, Gary Borzillo, Deborah Gordon, Elsa Barbacci-Tobin, Vincent Bernardo,David Briere, Tracey Clark, Matthew Corbett, John Jakubczak, Shefali Kakar, Elizabeth Knauth, Blaise Lippa,Michael J. Luzzio, Mahmoud Mansour, Gary Martinelli, Matthew Marx, Kendra Nelson, Jayvardhan Pandit,Francis Rajamohan, Shaughnessy Robinson, Chakrapani Subramanyam, Liuqing Wei, Martin Wythes, and Joel Morris

Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340

Received March 26, 2010

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from anelaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that wasmodestly selective and medicinal chemistry optimization of that lead to provide more selectiveanalogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer frompoor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiencyand the ability to balance overall druglike propeties with the careful control of lipophilicity in the leadseries. Discovery of the nonracemic amide series and subsequent modification produced an advancedanalogue that performed well in advanced preclinical assays, including xenograft tumor growthinhibition studies, and this analogue was nominated for clinical development.

Introduction

Akt (PKB)a is a serine/threonine kinase that plays a centralrole in promoting cell survival. Akt denotes a family of kinases(Akt1,Akt2,Akt3)with tissue-specificpatternsof expression thatcan include the coexpression of multiple family members in nor-mal tissues and tumors.1-3 Akt is part of a pathway that encom-passesmultiple receptor tyrosine kinases (PI3K andmTOR) andintegrates extracellular signalswith cell growth, proliferation, andsurvival. Akt activity is often deregulated in malignant cells, andevidence continues toaccumulate thatAkt is apromisingpointoftherapeutic intervention for many cancers.4,5 Akt is located at acritical junction of multiple oncogenic and tumor suppressionsignaling pathways. Deregulation of Akt through inactivation ofPTEN,6 pointmutation, or overexpression can result in aberrantsignaling.7 High levels of Akt activity are observed in a largenumberofhumancancersandarecorrelatedwithpoorprognosisand resistance to chemotherapy.8,9

Because it encompasses such promising targets for cancertreatment, the PI3K-mTOR pathway in general, and Aktspecifically, has generated substantial interest in the scientificcommunity.10,11Within the pharmaceutical industry,multiple

therapeutic approaches have been reported,12-14 including theuse of rapamycin and small molecule analogues as mTOR inhi-bitors,15-18 PI3K inhibitors,19,20 PI3K/mTOR dual inhibi-tors,21-23 and inhibitors that modulate Akt activity in both adirect ATP-competitive24-30 and an indirect (allosteric)31-38

fashion. Continued reports of tumor growth inhibition withAkt inhibitors in animal models reflect ongoing attempts tointerfere with this pathway and in some cases to include Aktinhibitors in combinationwithother treatment strategies.26,39,40

Results and Discussion

Our work started with high-throughput screening againstthe kinase domain of Akt1 in an effort to identify ATP-competitive inhibitors for further medicinal chemistry opti-mization. A description of this approach, which led to thediscovery of 1 and its subsequent optimization to 2 (Figure 1),was recently published.39

While spiroindoline 2 represented a substantial improvementinpotencyover the initial leads andan important investigationalcompound demonstrating tumor growth inhibition (TGI), ithad several undesirable properties. These included a distinctlackof selectivity againstmanyother kinases (particularly vs the

Figure 1. Aniline triazole 1 and spiroindoline 2.

†TheX-ray coordinates of compound 5bound toAkt1 andPKAhavebeen deposited in the Protein Data Bank with accession numbers 3MV5and 3MVJ. The X-ray coordinates of compound 42 bound to Akt1 havebeen deposited in the ProteinData Bankwith accession number 3MVH.

*To whom correspondence should be addressed. Phone: 858-526-4844. Fax: 860-686-5036. E-mail: [email protected].

aAbbreviations: ACN, acetonitrile; Akt, protein kinase B (encom-passing Akt1, Akt2, and Akt3); BOC, tert-butyl carbamate; DCM,dichloromethane; DIPEA, diisopropylethylamine; DMF, dimethylforma-mide; DMSO, dimethyl sulfoxide; GSK-3, glycogen synthase kinase 3;hERG, human ether-a-go-go-related gene; HLM Er, human liver micro-some extraction ratio; HOBt, 1-hydroxybenzotriazole; LAH, lithium alu-minum hydride; LipE, lipophilic efficiency; MeOH, methanol; mTor,mammalian target of rapamycin; PI3K, phosphoinositide 3-kinases;PKA, protein kinase A; PKB, protein kinase B (Akt); PTEN, phosphataseand tensin homologue; TEA, triethylamine; TFA, trifluoroacetic acid.

4616 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 Freeman-Cook et al.

highly homologous family member PKA) and a generally poorsafety profile in animal studies. This compound exhibiteddramatic effects on the GI tract and consequently had a verylowprojected therapeutic index (TI).An early hypothesis linkedits lowoverall kinase selectivitywith its lowTI, supportedby thefinding thata structurally similarbutkinase-inactivehomologueof2had significantly diminishedGI toxicity.41Thus, an effort todiscover new, more selective kinase inhibitors was initiatedutilizing high speed parallel chemistry.39

In this effort, potential new scaffolds were investigated byreplacing the spiroindoline substituent of 2 by thousands ofstructurally diverse amines. The data for selected analoguesare shown in Table 1.Most scaffolds were deliberately chosento incorporate basic primary or secondary amines that could

be utilized for further elaboration. In general, even whenpotent starting points (e.g., 6 and 7) were identified, theyexhibited a disappointing level of selectivity for Akt overPKA. In contrast, the unique selectivity displayed by the3-aminopyrrolidine series of analogues provided a compellingentry for further investigation.

The (3R)-aminopyrrolidine enantiomer 4 was both potentand19-fold selective forAktoverPKA.The (3S)-enantiomer 3,on the other hand, was somewhat less active (240 nM) anddisplayed equivalent activity against Akt and PKA. The factthat 5 displayed a similar level of selectivity to 4 suggested thatthe (3R)-aminopyrrolidine was responsible for conferring sel-ectivity, regardless of small modifications at C-5 of the corepyrrolopyrimidine. Compounds 2 and 5 were subjected tofurther broad kinase profiling, which demonstrated an overallhigh level of selectivity for compound 5, as shown in Table 2.

Our initial approach to elaboration of the (3R)-aminopyr-rolidine scaffold involved derivatization of the basic amine.By use of standard parallel chemistry transformations, amides,sulfonamides, and ureas were prepared; the amine was alsoalkylated via reductive amination reactions.

Disappointingly, all of these compounds, a sample of which isshown in Table 3, lost potency against Akt. More significantly,they exhibited reduced or reversed selectivity versus PKA. Theloss of potencyalonemaynot havebeen impossible toovercome;however, the difficulty in achieving selectivity against this targetin general argued that these modifications were disrupting animportant interaction, and thus, it would be extremely challen-ging to reconstruct a reasonable level of selectivity. Ultimately,crystal structures of 5 in the kinase domains of both Akt andPKA (Figure 2a and 2b, respectively) provided a structuralexplanation for the observed loss of selectivity.

TheX-ray structure of 5 in the active site ofAkt (Figure 2a)revealed that the C-3 primary amine is displayed in a

Table 1. New Scaffold Identificationa

aThe values are reported as the geometric mean of at least two separatedeterminations, with a typical standard variation of less than (30%. ThePKA selectivity ratio is calculated using the following formula: PKAselectivity = (PKA IC50)/(Akt IC50).

Table 2. Initial Kinase Selectivity Dataa

aValues shown are % inhibition data at 1 M, and reflect the mean ofat least two experiments.

Table 3. Amine substituent SAR expansiona

aThe values are reported as the geometric mean of at least two separatedeterminations, with a typical standard variation of less than (30%.

Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4617

pseudoaxial orientationand forms a salt bridge toGlu-234, aninteraction that would not be possible for the bulkier deriva-tized analogues. On the basis of this structural information,we proposed that introduction of substitution at the C-3methine would lock the amine into the required pseudoaxialconformation while simultaneously providing an additionalvector for optimization of binding potency.

Analysis of the crystal structure of PKA with 5 (Figure 2b)supported the hypothesis that thiswouldbe an effective strategyto maintain selectivity. Examination of the C-3 pyrrolidinemethine proton in the PKA structure indicated that any exten-sion at that position would likely result in a steric clash withnearby residues. Modeling the effect of adding even a singlemethyl group in place of the C-3 methine revealed a dramaticdecrease in predicted binding affinity for PKA, while thecorresponding binding inAkt was undisturbed. Together, theseobservations provided a compelling rationale to prepare theC-3substituted pyrrolidine analogues.

The approach to derivatization at the 3-position utilizedBOC-protected aminopyrroline 16 bearing a 3-hydroxy-methyl group (Scheme 1).42 Addition to chloropyrrolopyri-midine 15 proceeded smoothly. After oxidation and reductiveamination, the Boc was removed to reveal the elaboratedaminopyrrolidine analogues 18.

In the first set of compounds, anilines were installed duringthe reductive amination step (Table 4). Unsubstituted ani-line 19 was found to be similarly potent and selective com-pared to the original aminopyrrolidine 5, even when 19 wastested as a racemate. When 19 was separated into its two

enantiomers (19a and 19b), one was both more potent andmore selectivewhile the otherwas substantially poorer in bothrespects.43

Since the enantiomerically pure substituted 3-aminopyrroli-dine confirmed our structural design hypothesis, we undertook

Figure 2. X-ray structures of 5: (a, left) bound to Akt1; (b,right) bound to PKA.

Table 4. Initial SAR in the 3-Amino-3-Anilinomethylpyrrolidine Series

compd R chirality

Akt kinase

IC50

(nM)a

PKA

selectivity

ratio clogP LipE

HLM

Erb

19 -H rac 160 15.7 2.3 4.5 0.64

19a -H ent1 3500 2.7 2.3 3.2 0.75

19b -H ent2 56 22.7 2.3 5.0 ND

20 -Me rac 120 31.4 2.8 4.1 0.60

21 -Cl rac 58 37.5 3.3 3.9 0.61

22 -CF3 rac 61 20 3.7 3.5 0.75

23 -iPr rac 50 50.9 3.7 3.6 0.73

24 -OPh rac 16 51.2 4.4 3.4 0.73aThe values are reported as the geometric mean of at least two separate

determinations, with a typical standard variation of less than(30%. bEr isthe in vitro hepatic extraction ratio, which is obtained from dividingestimated hepatic blood clearance of test compounds by the human hepaticblood flow of 20 (mL/min)/kg. Protocols for measuring half-lives in HLMand further scaling to blood clearance have been published (see ref 44).

Figure 3. Pie charts showing the probability of success in higher logPspace: (A) HLM stability. Stable compounds (HLM Er < 0.5) arerepresented in green, and the overall percentages of stable compoundsare shown below the respective pie charts. (B) Dofetilide activity. Lowdofetilide activity compounds (Dof , <20% at 3 μM) are representedin green, and the overall percentages of compoundswith low dofetilideactivity are shown below their respective pie charts.

Scheme 1. Synthesis of 3,3-Disubstituted Analogues


the synthesis of additional analogues (Table 4). To illustrate atrend, only a subset of ortho-substituents is shown. Thesemodi-fications consistently showed improved potency as a result ofincreased lipophilicity.Unfortunately, theAkt potency gainwasoffset by a corresponding loss in human microsomal stabilityrelative to 3, a low clearance compoundwithHLMEr<0.30.44

Further examination of the data revealed that this was generallytrue in this series and not specifically related to the SAR of theortho-position of the aniline. Lipophilic substituents generallyprovided potency enhancements. However, the net effects ofincreased clearance and increased hERG binding45-47 madethese lipophilic analogues unattractive for further study. Ananalysis of the trends for in vitro clearance (Figure 3A) anddofetilide binding (Figure 3B), made evident by binning thedata, revealed that subsequent designs would likely need to betargeted at clogP< 3. At clogP>3 there was little chance ofachieving both low clearance and low hERG binding.

At this point, the focus changed from identifying the mostpotent compounds to identifying those compounds with themost activity per unit of lipophilicity (the highest LipE).48,49

Theoretically, these would provide the best starting points foroptimization with respect to the logP parameters outlinedabove and directly address multiple liabilities of this series.Additionally, consideration of LipE allows the identificationof truly beneficial SAR as opposed to potency gains that aresimply the consequence of large lipophilicity increases.

Modification of the synthetic route (Scheme 2) gave access tothe valuable amine intermediate 25 in racemic form. Additionof 3-pyrrolidinol to 4-chloro-5-methyl-7H-pyrrolopyrimidineproceeded smoothly in the presence of Hunig’s base. Oxidationof the resulting alcohol to the corresponding ketone with SO3-pyridine complex was followed by a modified Strecker reactionwith dimethoxybenzylamine using TMS-CN and ZnCl2 as aLewis acid.50 Reduction of the nitrile with lithium aluminum

hydride produced the valuable amine intermediate 25, whichcould be manipulated in a variety of standard transformations.To reduce the lipophilicity of the series, amide analogues wereprepared by coupling of carboxylic acids followed by TFA-mediated cleavage of the dimethoxybenzyl protecting group.

Acetamide26, prepared in racemic form(Table5), emergedasa notable analogue from this effort. This compound, while 300-foldweaker than derivatives that were being prepared in parallelin the aniline series, was a significant step forward in terms ofLipE (6.1 vs 4.5 for aniline 19). In contrast to compound 19, thehigh LipE of 26 coupled with its clogP of 0 suggested that thejudicious addition of lipophilicity within the amide frameworkcoulddrivean increase inpotencywhile still remainingwithin thedefined high probability space (clogP < 3).

The synthesis of 27 suggested that this concept was correct.The phenyl ring of 27 conferred a 10-fold potency improve-ment relative to the methyl substituent. This provided theimpetus to begin a full scale effort to optimize the amide inconjunction with the pyrrolopyrimidine C-5 substituent37

while maintaining an emphasis on lipophilic efficiency.We engaged in a focused incorporation of simple substitu-

ents expected to maintain clogP < 3. In particular, the orthoand para positions of the benzamide provided efficient loca-

tions to introduce substituents; thiswasmost apparentwith the

fluoro analogues 28, 29, and 30. In all cases, the fluorine

substituent provided an increase in potency relative to the

unsubstituted phenyl group of 27. In the ortho-position, this

improvement incurred no additional lipophilicity, while in the

para-position, the increase in potency was large enough to

compensate for the heightened lipophilicity. The higher LipE

values for both 28 and 30 reflect the role of the ortho- andpara-

positions in potency improvement for this series. The small

decrease in LipE for 29 versus 27 indicated that the meta-position was potentially less promising for manipulation.

Scheme 2. Modified Synthesis for Preparation of Racemic Amides

Table 5. SAR of Racemic Amide Analoguesa

compd R substituent substituent position

Akt kinase

IC50 (nM) clogP LipE HLM Er

26 Me none 765 0 6.1 ND

27 Ph none 62 1.7 5.5 ND

28 Ph -F ortho 44 1.6 5.7 0.39

29 Ph -F meta 38 2 5.4 0.39

30 Ph -F para 7.4 2 6.1 0.46aThe values are reported as the geometric mean of at least two separate determinations, with a typical standard variation of less than (30%.


Once this scan of substituents was complete, sets of com-pounds were prepared with enantiomerically pure amino-pyrrolidines51 using multiple pyrrolopyrimidine C-5 corevariations (-CN, -Cl, -Me, -Et). Again, a key aspect ofthe design was to keep the clogP below 3. For a given amide,the trends for potency, selectivity, and in vitro clearance arevery consistent (Table 6). Use of a polar core substituent(-CN) at C-5 reduced the logP and provided good micro-somal stability but attenuated the potency (entries 31, 35,and 39). Compromised potency combined with an apparentpermeability issue (in part related to the higher total polarsurface area) gave weaker cellular activity for these com-pounds in an assay measuring the phosphorylation of aknown Akt substrate, GSK-3. As the core substituents be-came more lipophilic (-Me, -Cl, -Et), potency improvedand stability was diminished. For benzamide substitution, the4-Cl analogues (35-38) all had lower lipophilic efficiencycompared with the corresponding 4-F compounds (31-34),similar to the results observed in the racemic series. Of thesubstituents examined, the 2,4-difluoro analogues (39-42)exhibited the best overall potency and metabolic profile.

All of the compounds in Table 6 displayed good selectivityfor PKA, and some had extraordinary selectivity. Particularlynotablewas compound 42, which achieved 900-fold selectivityfor Akt over PKA, presumably because of the increased stericdemand of the ethyl core substituent being better tolerated inAkt than in PKA.This, in conjunctionwith the beneficial effectof the 2,4-difluoro substitution forAkt, providedanextremelyselective analogue. After submission of 42 to a panel of 226kinases, only seven (CDK7, MSK1, MSK2, PKG1R, RSK2,RSK3, and PKA) showed >50% inhibition at 1 μM. Afterdeterminination of IC50 values, 42 demonstrated >100-foldselectivity against Akt for all seven kinases.52 Cocrystallizationof 42withAkt (Figure 4) showed that theC-3 aminewas enga-ged in the Glu-234 salt bridge, similar to the early analogues.As expected, the 2,4-difluorobenzamidewas extended into theaccessible pocket in Akt.

On the basis of these in vitro data, a small set of these ad-vanced analogues were put into further studies, including ratand dog pharmacokinetic studies, as well as in vivo models of

xenograft tumor progression.On the basis of these data, 42waschosen as the most promising molecule to advance to clinicaldevelopment. In vitro, 42 proved highly permeable, and in dogpharmacokinetic studies (oral at 2 mg/kg, iv at 1.2 mg/kg) thecompound was well absorbed (F = 54%), with moderateclearance (11.6 (mL/min)/kg) and volume of distribution(4.8 L/kg), and a half-life of 4.4 h. Compound 42 was subse-quently evaluated for modulation of Akt in tumors53 and inmultiple in vivo mouse models of antitumor efficacy. It wasactive in a PC3 prostate carcinoma xenograft experiment, with75%TGI observed at 100mg/kg b.i.d. dosing for 10 days. In acolorectal carcinoma (Colo205) xenograft study, 42 produced60% TGI at 150 mg/kg b.i.d. after 10 days. Most intriguingly,in combination with rapamycin (10 mg/kg, ip), 75 mg/kg b.i.d.(10 days) of 42 resulted in 98% TGI in an additional PC3prostate carcinomaxenograft study compared to56%TGIand66% TGI with 42 and rapamycin as single agents. There wereno significant toxicological side effects observed in these studiesbeyond modest weight loss in the highest dosing groups.

Conclusion

In summary, we have outlined the optimization of 2-4 toadvanced molecules such as 42 using a combination of both

Table 6. SAR of Nonracemic (3S)-Amino-aminomethylbenzamide Analoguesa

compd R Ph substituent

Akt kinase

IC50 (nM)

Akt cell

IC50(μM) PKA selectivity ratio LipE HLM Er clogP

31 CN 4-F 7.8 5.74 120.3 7 0.26 1.1

32 Me 4-F 3.0 1.46 522 6.5 0.36 2

33 Cl 4-F 2.6 0.36 115.4 6.3 0.55 2.3

34 Et 4-F 1.4 0.65 645 6.3 0.63 2.6

35 CN 4-Cl 17.5 6.34 ND 6.1 0.38 1.6

36 Me 4-Cl 11.4 1.95 51.8 5.3 0.62 2.6

37 Cl 4-Cl 3.3 0.35 39.8 5.6 0.61 2.9

38 Et 4-Cl 1.8 0.92 ND 5.6 0.67 3.1

39 CN 2,4-diF 7.7 1.86 88.4 7.3 0.26 0.9

40 Me 2,4-diF 1.0 0.31 ND 7.2 0.39 1.8

41 Cl 2,4-diF 1.1 0.28 50.9 6.9 0.47 2.1

42 Et 2,4-diF 0.5 0.31 900 6.9 0.56 2.4aThe kinase and cell values are reported as the geometricmean of at least two separate determinations, with a typical standard variation of less than(30%.

Figure 4. Cocrystal structure of 42bound to theAkt1 kinase domain.


structure- and physical-property-based design parameters.Substituents in place of the C-3 methine proton of the amino-pyrrolidine lead 4 served to lock the pseudoaxial disposition ofthe amine and to provide a platform for optimizing the physicalproperties of the resulting molecules. Critical in this effort wasthe understanding of overall lipophilicity and its effect on bothpotency and in vitro ADME properties. During lead optimiza-tion, changes in LipE highlighted valuable structural modifica-tions and discouraged the overuse of lipophilicity, which ulit-mately providedmore balancedmolecules. Compound 42 pro-vided significantly enhanced selectivity for Akt relative toearlier leads such as spiroindoline 2. As a result, 42 was nomi-nated for clinical development and represents a broadly selec-tive, potent, ATP-competitive Akt inhibitor.

Experimental Section

General Experimental Details.All solvents and reagents wereobtained from commercially available sources and used withoutfurther purification. Reactions were carried out under nitrogenatmosphere unless otherwise described. Flash chromatographypurifications were performed on prepacked silica gel cartridgeson ISCO CombiFlash Companion systems using the indi-cated mobile phases. HPLC analysis was performed on anAgilent HP1100 system with an Agilent Zorbax SB-CN column(4.6 mm� 75 mm, 3.5 μm, 1.5 mL/min flow rate), mobile phase(H2O/0.1% HClO4)/(ACN/0.1% HClO4) (90:10 at 0 min to10:90 at 5 min). All tested compounds prepared as solids weredetermined to be>95%pure at 210 nm. LC/MSSTD refers to aPolaris C18A column (2.0 mm� 20mm, 5 μm, 1.0 mL/min flowrate); mobile phase A of 94.95%H2O, 5% CAN, 0.05% formicacid; mobile phase B of 99.95% CAN, 0.05% formic acid; A/Bratios of 95:5 at 0min, 80:20 at 1min, 50:50 at 2.3min, and 0:100at 3.6 min, with compound detection by UV and MS. LC/MSPolar refers to a Polaris C18A column (2.0 mm� 20 mm, 5 μm,1.0 mL/min flow rate), mobile phase A of 94.95% H2O, 5%ACN, 0.05% formic acid; mobile phase B of 99.95% ACN,0.05% Fformic acid; A/B ratios of 95:5 at 0 min, 80:20 at 2 min,50:50 at 2.3 min, and 0:100 at 3.6 min, with compound detec-tion by UV and MS. Mass spectral data were collected on aMicromass ADM atmospheric pressure chemical ionizationinstrument (LRMS APCI). NMR spectra were generated ona Varian 400 MHz or a Varian 500 MHz instrument as indi-cated. Chemical shifts were recorded in ppm relative to tetra-methylsilane (TMS) with multiplicities given as s (singlet), bs(broad singlet), d (doublet), t (triplet), dt (double of triplets), m(multiplet).

Assay Description. Akt1 Kinase Assay.A fluorescence polar-ization IMAP type assay is used. An amount of 15 μL of dilutedtest compound in DMSO is mixed with 60 μL of reaction buffer(10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EGTA,0.01% Triton-X100, 1 mMDTT). Then 5 μL of the compound/buffer mixture, 10 μL of a solution containing 4 μM ATP and40 nM fluorescent-labeled Crosstide (Tamara-labeled GRPR-TSSFAEG peptide), and 5 μL of Akt1 protein (lacking thepleckstrin homology (PH) domain, containing an Asp at posi-tion 473, and prephosphorylated at Thr 308) in reaction bufferare combined. After a 90 min incubation, IMAP beads areadded and plates are read (lamp filter, 544 nm; emission filter,615 nm). The same procedure can be applied to full length Akt1to provide similar results. All IC50 values are the geometricmeanof at least n = 2 determinations.

PKAKinase Assay.A fluorescence polarization IMAP (Mole-cular Devices Corp.) type assay is used. An amount of 15 μL ofdiluted test compound in DMSO is mixed with 60 μL of reactionbuffer (10mMTris-HCl, pH 7.5, 10mMMgCl2, 0.1mMEGTA,0.01% Triton-X100, 1 mMDTT). Then 5 μL of the compound/buffer mixture, 10 μL of a solution containing 4 μM ATP and40nMfluorescent-labeledKemptide (Tamara-labeledLRRASLG

peptide), and 5 μL of recombinant PKA catalytic subunit (Up-state Biotechnology, catalog number 14-440) in reaction bufferare combined. After a 90 min incubation, IMAP beads areadded and plates are read (lamp filter, 544 nm; emission filter,615 nm). All IC50 values are the geometric mean of at least n=2determinations.

Akt Cell Assay. The phosphorylation status of an Akt sub-strate, GSK-3R, in the U87 glioblastoma line was measured asan indicator of cellular activity. U87 cells (10 000) were platedovernight in DMEM medium containing 15% fetal calf serum(FCS) and incubatedwith compound (seven concentrations plusvehicle) for 2 h in DMEMmedium lacking FCS. After cell lysis,45%of the lysate was transferred to a 96-well, flat bottom, blackpolystyrene plate (Costar, catalog number 3295) previouslycoated in a stepwise fashion with 100 μL/well of goat antirabbitIgG (Pierce Corp., catalog number 31210) diluted to 5 μg/mL incarbonate-bicarbonate buffer (Sigma, catalog number C-3041),followed by TBST washing (Sigma, catalog number T-9039),blocking in 5% BSA/TBST, and addition of 100 μL/well of aphosphospecific antibody to GSK-3R/β (Cell Signaling Tech-nology, catalog number 9331) used at 0.06 μg/mL. After sub-sequent washing and blocking steps, cell lysate was added andincubated for 1 h at ambient temperature with shaking. Theplates were then washed four times with TBST. To detect boundphospho-GSK-3R, 70 μL of a 1:1000 dilution of anti-GSK-3Rwas added (Santa Cruz, catalog number SC-5264). Plates wereincubated for 1 h at ambient temperature with shaking, andwashed four times with TBST. After washing, 70 μL/well of a1:5000 dilution of goat antimouse-HRP (Jackson Labs, catalognumber 115-035-146) in 5%BSA/TBST was added. Plates wereincubated for 1 h at ambient temperature with shaking, washed,and 70 μL/well of SuperSignal ELISA PICO Maximum Sensi-tivity Substrate (Pierce Corp., catalog number 37075) wasadded. Signal was allowed to develop for 1 min, and plates wereread on a Victor luminometer. The signal obtained from wellscontaining lysis buffer only (no cell control) was subtractedbefore IC50 values were calculated. All IC50 values are thegeometric mean of at least n = 2 determinations.

1-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3((phenylamino)-methyl)pyrrolidin-3-amine (19). A mixture of aniline (0.16 mL,1.77 mmol) and 3 A molecular sieves (0.5 g) was added to asolution of tert-butyl 3-formyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyri-midin-4-yl)pyrrolidin-3-ylcarbamate 17 (0.5 g, 1.45 mmol),glacial acetic acid (1 mL), and MeOH (9 mL). The resultingreaction mixture was stirred at 25 �C overnight, treated withMP-cyanoborohydride (1.45 g, 2.5 mmol/g, 3.65 mmol), andstirred for an additional 5 h. The mixture was filtered, and thesolids were rinsed with MeOH. The combined organic phaseswere concentrated to provide tert-butyl 1-(5-methyl-7H-pyrrolo-[2,3-d]pyrimidin-4-yl)-3-((phenylamino)methyl)pyrrolidin-3-yl-carbamate, which was treated directly with DCM (10 mL) andTFA (10 mL), and the resultant reaction mixture was stirred at25 �C for 3 h. Themixture was concentrated, and the residuewaspurified by chromatography on silica gel (eluting with dichlor-omethane, with a gradient ramp to 1% aqueous NH4OH, 29%MeOH, 70% DCM) to provide 1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3-((phenylamino)methyl)pyrrolidin-3-amine(19). Yield: 325 mg, 1.01 mmol, 70%. APCI MSþ (M þ 1)=323.2. 1H NMR (500 MHz, methanol-d4) δ ppm 2.03 (1H, m),2.21 (1H, m), 2.38 (3H, s), 3.37 (2H, m), 3.68 (1H, d), 3.89 (1H,d), 3.91(1H, m), 4.05 (1H, q), 6.60(1H, t), 6.69 (2H, d), 6.92 (1H,s), 7.08 (2H, m), 8.08 (1H, s).

N-((3-Amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-pyrrolidin-3-yl)methyl)benzamide (27). A solution of 3-(amino-methyl)-N-(2,4-dimethoxybenzyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-amine (50 mg, 0.13 mmol) inDMF (1.2 mL) was treated with 1-hydroxybenzotriazole (HOBt)(26 mg, 0.19 mmol), benzoic acid (16 mg, 0.13 mmol), and PS-carbodiimide (160 mg, 0.25 mmol). The resultant reaction mixturewas stirred at 25 �C for 6 h, treated with MP-carbonate (160 mg,


0.5 mmol), and stirred overnight. The mixture was filtered, and thesolids were rinsed withMeOH. The combined filtrates were evapo-rated, and the residuewas treatedwith TFA (0.5mL) and heated at80 �C for 3 h. The reaction mixture was concentrated, and theresidue was purified by preparative RP-HPLC (TFA/ACN/H2O)to provideN-((3-amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-3-yl)methyl)benzamide (27). Yield: 12 mg, 26% fortwo steps.MSþ (Mþ 1)þ: 351.3. tR (LCMS standard): 0.7min. 1HNMR (400 MHz, methanol-d4) δ ppm 8.03 (s, 1 H), 7.82 (d, J =7.48Hz,2H), 7.53 (t,J=7.06Hz, 1H), 7.40-7.49 (m,2H), 6.90 (s,1H), 3.85-4.05 (m, 3H), 3.54-3.68 (m, 3H), 3.31 (s, 2H), 2.39 (s,3 H), 2.08-2.19 (m, 1 H), 1.92-2.01 (m, 1 H).

N-((3-Amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyr-rolidin-3-yl)methyl)-4-fluorobenzamide (30). Compound 30 wasprepared by an identical sequence of reactions as was used toprepare 27 but using 4-fluorobenzoic acid in place of benzoicacid. 1HNMR (DMSO-d6) δ ppm 11.3 (bs, 1H), 8.56 (t, J=5.7Hz, 1H), 8.04 (s, 1H), 7.94 (dd, J = 8.8, 5.7 Hz, 2H), 7.30 (dd,J=8.8, 8.8Hz, 2H), 6.93 (s, 1H), 3.88-3.82 (m, 1H), 3.75-3.71(m, 1H), 3.68 (d, J = 10.9 Hz, 1H), 3.47 (m, 3H), 2.31 (s, 3H),1.95-1.73 (m, 4H).

(S)-N-((3-Amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-pyrrolidin-3-yl)methyl)-2,4-difluorobenzamide (42).Amixture of(R)-N-((3-aminopyrrolidin-3-yl)methyl)-2,4-difluorobenzamide(16 g, 49 mmol), 4-chloro-5-ethyl-7H-pyrrolo[2,3-d]pyrimidine(8.9 g, 49 mmol), and sodium bicarbonate (20.5 g, 244 mmol) inethanol (150 mL) was refluxed for 10 h. The mixture was thenfiltered hot through Celite, and the filtrate was concentratedunder reduced pressure. The resultant residue was partitionedbetween EtOAc (100 mL) and water (200 mL), and the organicphasewas collected. The aqueous phasewas extractedwith ethylacetate (2 � 100 mL), and the combined organic extracts werewashed with water (2� 100mL), brine (80mL), dried over Na2-SO4, filtered, and concentrated under reduced pressure toprovide (S)-N-((3-amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimi-din-4-yl)pyrrolidin-3-yl)methyl)-2,4-difluorobenzamide (42) asa pale-yellow solid. Yield: 17.4 g, 89.3% . TLC Rf = 0.42 (10%methanol/CH2Cl2). APCI MSþ (M þ 1) = 402.1. 1H NMR(500 MHz, methanol-d4) δ ppm 8.07 (s, 1 H), 7.80 (q, J = 8.64Hz, 1 H), 7.10 (q, J=8.12 Hz, 2 H), 6.94 (s, 1 H), 3.98 (m, 3 H),3.65 (m, 3H), 2.87 (q, J=7.08Hz, 2H), 2.17 (m, 1H), 1.97 (m, 1H), 1.30 (t, J = 7.26 Hz, 3 H).

Acknowledgment. The authors want to thank KatherineBrighty for extremely helpful advice in manuscript prepara-tion and editing.

Supporting Information Available: Experimental details forkey intermediates and for compounds 31-41; details of theX-ray crystallography and pharmacokinetic and pharmacody-namic studies. This material is available free of charge via theInternet at http://pubs.acs.org.

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design of selective, atp-competitive inhibitors of akt

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